Species of Bacillus, such as Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, are attractive microorganisms for recombinant protein production in view of their fast growth rate, high yield, and ability to secrete produced products directly into the medium. Bacillus anthracis is also attractive in view of its ability to produce anthrax toxin and ability to fold proteins correctly. However, the attractiveness of Bacillus is reduced in view of the large quantities of extracellular proteases that the microorganisms secrete into the medium, leading to protein degradation, and the fact that they form spores.
The Gram-positive bacterial pathogen Bacillus anthracis (also referred to herein as B. anthracis) secretes high levels of the three proteins that are collectively termed anthrax toxin: protective antigen (PA), edema factor (EF), and lethal factor (LF), when grown under conditions thought to mimic those in an infected animal host. PA is a receptor-binding component which acts to deliver LF and EF to the cytosol of eukaryotic cells; EF is a calmodulin-dependent adenylate cyclase, and LF is a zinc metalloprotease that cleaves most members of the mitogen-activated protein kinase kinase family; see, for example, Leppla S H et al., 2000, in Anthrax toxin, bacterial protein toxins, 445-472, Aktories K et al., (Eds.), Springer, Berlin; Moayeri M et al., 2011, Anthrax toxins, Bacillus anthracis and Anthrax, 121-156, Bergman (Ed.), John Wiley & Sons, Inc., Hoboken, N.J.; Moayeri M et al., 2009, Mol. Aspects Med. 30, 439-455; Young JA et al., 2007, Annu. Rev. Biochem. 76, 243-265. PA, EF, and LF are encoded on virulence plasmid pXO1 by pag, cya, and lef, respectively. Virulence plasmid pXO2 encodes proteins required for capsule formation and depolymerization. B. anthracis that lack one or both virulence plasmids are typically attenuated in most animal hosts. Single PA, EF, and LF components are non-toxic; a combination of one PA and at least two EFs, at least two LFs, or mixtures of EF and LF are required for toxicity.
Because anthrax pathogenesis is highly dependent on the actions of the anthrax toxin proteins, vaccine and therapeutic development efforts have focused on countering toxin action, typically by generating antibodies to PA. The anthrax vaccine currently licensed in the USA, and developed almost 50 years ago (see, e.g., Puziss M et al., 1963, J. Bacteriol. 85, 230-236), consists of a partially purified culture supernatant of a protease-deficient B. anthracis strain (V770-NP1-R). PA is the most abundant protein and the key immunogen in this vaccine. Efforts to produce a recombinant PA vaccine from B. anthracis by scale-up of an established process (see, e.g., Farchaus JW et al., 1998, Appl. Environ. Microbiol. 64, 982-991) appear to have been hampered by instability of the final product.
While the toxin components can be purified as recombinant proteins from B. anthracis culture supernatants (see, e.g., Farchaus JW et al., ibid.; Varughese M et al., 1999, Infect. Immun. 67, 1860-1865; Singh Y et al., 1991, J. Biol. Chem. 266, 15493-15497; Park S et al., 2000, Protein Expr. Purif. 18, 293-302), the integrity and yields are limited by the B. anthracis proteolytic enzymes that are co-secreted.
Two extracellular proteases are reported to be abundant in the B. anthracis secretome: NprB (GBAA_0599), neutral protease B, a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species; and InhA1 (GBAA_1295), immune inhibitor A1, a homolog of the immune inhibitors A from other members of the Bacillus cereus group (see, e.g., Antelmann H et al., 2005, Proteomics 5, 3684-3695; Chitlaru T et al., 2006, J. Bacteriol 188, 3551-3571; Chung MC et al., 2006, J. Biol. Chem. 281, 31408-31418. These two proteases contain zinc-binding motifs typical for the zincin tribe of metallopeptidases (His-Glu-Xxx-Xxx-His (SEQ ID NO:31)) and belong, respectively, to the M4 and M6 families of metalloproteases according to the MEROPS database, Wellcome Trust Sanger Institute (see e.g., website of Wellcome Trust Sanger Institute).
A third metalloprotease, camelysin (GBAA_1290), belonging to the M73 family is found in the secretome of several B. anthracis strains. This protease is similar to the camelysin of B. cereus, a novel surface metalloprotease; see, e.g., Grass G et al, 2004, Infect. Immun. 219-228.
B. anthracis also contains a gene encoding InhA2 metalloprotease (GBAA_0672, M6 family), although it is not known whether this protease is expressed and secreted. This gene is an ortholog of the InhA1 described above (68% amino acid identity). Similarly, the genome of B. anthracis also contains genes encoding TasA (GBAA_1288, M73 superfamily), which is an ortholog of camelysin (60% amino acid identity), and MmpZ (GBAA_3159, ZnMc superfamily), which is a putative extracellular zinc-dependent matrix metalloprotease, a member of the metzincin clan of metallopeptidases. This clan is characterized by an extended zinc-binding motif (His-Glu-Xxx-Xxx-His-Xxx-Xxx-Gly/Asn-Xxx-Xxx-His/Asp (SEQ ID NO:32)) (see, e.g., Gomis-Ruth, FX, 2009, J. Biol. Chem. 284, 15353-15357).
Bacillus subtilis strains having more than one protease inactivated have been produced and analyzed. For example, Wu X C et al., 1991, J. Bacteriol. 173, 4952-4958 produced a B. subtilis strain deficient in six extracellular proteases (WB600), namely neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B. WB600 showed only 0.32% of the extracellular protease activity of wild-type B. subtilis strains. Kurashima K et al., 2002, J. Bacteriol. 184, 76-81, expressed apparently intact Clostridium cellulovorans EngB cellulase in a B. subtilis strain deficient in eight proteases (WB800). WB800 was derived from WB600 through inactivation of VpR protease and cell wall protease WprA. WB700 was derived from WB600 through inactivation of VpR (see, e.g., Wu et al, 2002, Appl. Environ. Microbiol. 68, 3261-3269).
There have been reports of inactivation of certain individual B. anthracis proteases: Inactivation of B. anthracis NprB led to reduced proteolysis of casein (see, e.g., Pomerantsev A P et al., 2006, Infect. Immun. 74, 682-693. Inactivation of InhA1 indicated that coagulation of human blood by B. anthracis required InhA1 for proteolytic activation of prothrombin and factor X (see, e.g., Kastrup C J et al., 2008, Nat. Chem. Biol. 4, 742-750. However, production of anthrax toxin proteins in both of these strains led to protein degradation over time, albeit at a later time than production in B. anthracis A35.
There remains a need for a B. anthracis that can produce large amounts of stable (i.e., intact) proteins, such as anthrax toxin proteins PA, EF, and LF.